AUTOREACTIVE ANTIGENS IN BEHCET DISEASE WITH PROTEIN MACROARRAY METHOD

Elcin SEHITOGLU, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul Filiz CAVUS, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul Elif UGUREL, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul L Sukru ATAKAN, Department of Molecular Biology and Genetics, Bilkent University, Ankara

Erdem TUZUN, Department of Neuroscience, Institute for Experimental Medicine (DETAE), Istanbul

Ahmet GUL, Division of Rheumatology Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul

Gülsen Akman DEMIR, Neurology Department, Medical Faculty of Istanbul, Istanbul

Can Fahrettin KOYUNCU, Department of Computer Engineering, Bilkent University, Ankara Çiğdem Gündüz DEMIR, Department of Computer Engineering, Bilkent University, Ankara Ali Osmay GURE, Department of Molecular Biology and Genetics, Bilkent University, Ankara Ugur OZBEK, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul Burcak VURAL, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul Behçet\'s disease (BD) is a chronic, recurrent and inflammatory disorder characterized with oral and genital aphthous ulcerations, uveitis, skin lesions and skin pathergy reaction. Although the etiology of BD remains unknown, the presence of inflammatory lesions and identification of antibodies directed against antigens shared between microorganisms and the involved tissues of the patients [e.g. heat shock proteins (HSPs)] have suggested an autoimmune nature. In this study, we aimed to determine disease spesific antigens in serum samples of BD patients with Protein Macroarray Method, then identify antibodies sensitivity/spesificity against to these autoantigens with enzyme-linked immunosorbent assay (ELISA) and Western Blot. Serum samples of BD patients (n=101) with arthritis involvement (n=28), mucocutaneous involvement (n=26), uveitis (n=19) and vascular involvement (n=23); healthy controls (HC) (n=100) without an autoimmune or inflammatory disease were studied. Pool of BD patients and HC were screened by using a high-density protein macroarray derived from human fetal brain cDNA expression library (hEX1- ImaGenes, Berlin, Germany). To identify the seroreactivity of antibodies arrays were prepared and incubated with pooled serum samples from each study group according to manufacturer’s recommendation. Images were capture then analyzed and 12 most reactive antigens were selected. Selected positive clones (FAM32A, GON4L, ANAPC5, SARNP, RPL18, B-cell lymphoma/leukemia 11A, CKB, JUP, FTH1, TCEA2, STMN4, CCNL2) (ImaGenes) were obtained, His6-tagged proteins were recombinantly expressed in E. coli and purified by affinity chromatography. Purified proteins will use for ELISA and Western Blot with BD patients serums and HC serums in further analysis. ELISA results will statistically evaluate with ANOVA as a result of BD patients compare with HC. The results of this study might enable determination of antibodies that are potentially involved in BD pathogenesis. Some of these antibodies with high sensitivity and specificity to BD might be used as biomarkers in the future.

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PEPTIDE-BASED MONOCLONAL ANTIBODY PRODUCTION AGAINST β-ACTIN PROTEIN

Nazila AMINI, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran

Mohadeseh NAGHI VISHTEH, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran

Sayeh KHANJANI, Reproductive biotechnology Research Center, Avicenna Research Institute, Tehran

Forough TORABI, Reproductive biotechnology Research Center, Avicenna Research Institute, Tehran

Omid ZAREI, Department of Pharmaceutical Biotechnology,Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz

Reza HADAVI, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran Hodjattallah RABBANI, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran

Mahmood JEDDI-TEHRANI, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran

Aim: Actin is one of the most widely studied structural and multifunctional proteins in eukaryotic cells that has important roles in many cell functions. The aim of this study was producing monoclonal antibodies against a synthetic peptide derived from N-terminal region of β-actin protein in order to use in different applications. Methods: A synthetic peptide derived from β-actin was designed and used to immunize two Balb/c mice. The mice were bled and serum ELISA assay was performed to evaluate the immunization efficiency. In order to produce hybridoma cells, spleen of the immunized mouse with higher serum antibody titer was removed at sterile conditions and spleenocytes were fused to mouse myeloma SP2/0 cell line followed by seeding into 96-well plates in the presence of selective HAT medium. Clone formation was examined daily and the presence of antibody against the immunizing peptide was determined by ELISA. The produced antibody was purified from supernatant of cultured selected-clones by affinity chromatography column prepared by coupling the immunizing peptide to CNBr-activated sepharose 4B . The quality of the purified antibody was evaluated by SDS-PAGE. Its specific interaction with the immunizing peptide was determined by ELISA. Immunoreactivity of the antibody was also tested by immunocytochemistry (ICC) as well as immunohistochemistry (IHC) and Western blotting (WB) in a panel of organisms with different origins. All experiments were repeated using a commercial antibody for comparison. Results: The results showed that the antibody had desired purity and reactivity with the peptide-BSAconjugate in SDS-PAGE and ELISA, respectively. It could also detect a band of about 45 kDa corresponding to β-actin protein in WB. Moreover, the antibody could react with β actin in ICC and IHC. Conclusion: The anti-β-actin antibody may be used as an internal control for WB and may serve as a tool in different experiments such as ELISA, ICC and IHC.

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Keywords: Actin Monoclonal Antibody Western blotting Immunocytochemistry

Immunohistochemistry

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Western blot of different samples and produced antibody

Table of Western blot, Immunocytochemistry and Immunohistochemistry

Sample name Western blot Immunohistochmeistry or

Oyster + +

Crab - -

Neon Fish - -

Daphnia + -

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EVALUATION OF PAX5 GENE IN THE EARLY STAGES OF LEUKEMIC B CELLS IN THE